U.S. patent number 5,756,680 [Application Number 08/678,364] was granted by the patent office on 1998-05-26 for sequential separation of whey proteins and formulations thereof.
This patent grant is currently assigned to Sepragen Corporation. Invention is credited to Salah H. Ahmed, Quirinus R. Miranda, Zahid Mozaffar, Vinit Saxena.
United States Patent |
5,756,680 |
Ahmed , et al. |
May 26, 1998 |
Sequential separation of whey proteins and formulations thereof
Abstract
A method is disclosed for the sequential separation of whey
proteins using radial-flow chromatography. Different buffer systems
adjusted to suitable pH and ionic strength are utilized in the
separation process. The method separates at least five different
proteins from whey. Infant feeding formulas, and other food
formulations are also disclosed incorporating therein in different
proportions various proteins separated from the whey.
Inventors: |
Ahmed; Salah H. (Hayward,
CA), Saxena; Vinit (Pleasanton, CA), Mozaffar; Zahid
(Union City, CA), Miranda; Quirinus R. (San Jose, CA) |
Assignee: |
Sepragen Corporation (Hayward,
CA)
|
Family
ID: |
22649135 |
Appl.
No.: |
08/678,364 |
Filed: |
July 16, 1996 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
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177574 |
Jan 5, 1994 |
|
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Current U.S.
Class: |
530/366; 426/271;
426/41; 426/583; 530/350; 530/364; 530/386; 530/394; 530/414;
530/416 |
Current CPC
Class: |
A21D
2/263 (20130101); A23C 9/1465 (20130101); A23J
1/205 (20130101); C07K 16/04 (20130101); A23L
21/15 (20160801); A23L 33/19 (20160801); A23V
2002/00 (20130101); G01N 2030/386 (20130101); A23V
2002/00 (20130101); A23V 2250/54242 (20130101); A23V
2250/54244 (20130101); A23V 2250/54248 (20130101); A23V
2250/5425 (20130101); A23V 2300/30 (20130101) |
Current International
Class: |
A23C
9/146 (20060101); A23C 9/00 (20060101); A23J
1/00 (20060101); A23J 1/20 (20060101); A23L
1/06 (20060101); A23L 1/068 (20060101); A23L
1/305 (20060101); A21D 2/00 (20060101); A21D
2/26 (20060101); C07K 16/04 (20060101); G01N
30/38 (20060101); G01N 30/00 (20060101); C07K
016/04 (); C07K 014/47 (); C07K 001/36 (); A23C
009/14 () |
Field of
Search: |
;530/350,363,364,365,366,386,394,412,414,416,832
;426/41,271,583 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Girardet et al. "Effects of Chromatographic Parameters on the
Fractionation of Whey Proteins by Anion Exchange
FPLC"Milchwissencharft 44(11) 692-696 1989. .
H.o slashed.st et al. "Prospective Estimation of lgG,IgG Subclass
& IgE Antibodies to Dietary Proteins In Infants w/Cow Milk
Allergy"Allergy 47 (3) 218-229 1992 .
Manji et al. "Rapid Separation of Milk Whey Proteins by Anion
Exchange Chromatography"J. Dairy Sci 68 3176-3179 1985..
|
Primary Examiner: Fleisher; Mindy
Assistant Examiner: Degen; Nancy J.
Attorney, Agent or Firm: Medlen & Carroll
Parent Case Text
This is a continuation of application Ser. No. 08/177,574 filed on
Jan. 5, 1994, now abandoned.
Claims
What is claimed is:
1. A method for the sequential separation of whey proteins,
comprising the steps of:
a) packing a chromatographic column with a cationic exchange resin
to provide a packed chromatographic column;
b) equilibrating said packed chromatographic column with a
buffer;
c) providing a whey sample containing whey proteins comprising
lactoferrin, immunoglobulin, -lactoglobulin, -lactalbumin, and
bovine serum albumin;
d) passing said whey sample through said packed chromatographic
column under conditions whereby at least a portion of said whey
proteins adsorb to said packed chromatographic column;
e) washing said packed chromatographic column with a buffer;
f) sequentially eluting immunoglobulin and -lactoglobulin from said
packed chromatographic column with a buffer;
g) reconditioning said packed chromatographic column with a
buffer;
h) eluting -lactalbumin from said packed chromatographic column
with a buffer;
i) reconditioning said packed chromatographic column with a
buffer;
j) eluting bovine serum albumin from said packed chromatographic
column with a buffer; and
k) eluting lactoferrin from said packed chromatographic column with
a buffer to create an eluate containing lactoferrin.
2. The method of claim 1, wherein said whey is selected from the
group consisting of pasteurized sweet whey, pasteurized acid whey,
non-pasteurized acid whey, and whey protein concentrate.
3. The method of claim 1, wherein said chromatographic column is a
radial flow column.
4. A method for the separation of -lactoglobulin from whey
proteins, said method comprising the steps of:
a) packing a radial flow chromatographic column with an anion
exchange resin to provide a first packed chromatographic
column;
b) equilibrating said first packed chromatographic column with a
buffer;
c) providing a whey sample containing whey proteins comprising
lactoferrin, immunoglobulin, -lactoglobulin, -lactalbumin, and
bovine serum albumin;
d) passing said whey sample through said first packed
chromatographic column under conditions wherein -lactoglobulin
adsorbs to said first packed chromatographic column and a permeate
flows through said first packed chromatographic column;
e) collecting said permeate from said first packed chromatographic
column, wherein said permeate comprises -lactalbumin,
immunoglobulin, bovine serum albumin and lactoferrin;
f) eluting said adsorbed -lactoglobulin from said first packed
chromatographic column with a buffer to produce an eluate;
g) packing a second chromatographic column with a cation exchange
resin to provide a second packed chromatographic column;
h) equilibrating said second packed chromatographic column;
i) passing said permeate from said first packed chromatographic
column through an ultrafiltration membrane to produce an
ultrafiltrate;
j) passing said ultrafiltrate through said second packed
chromatographic column, under conditions such that immunoglobulin,
-lactalbumin, bovine serum albumin and lactoferrin adsorb to said
second packed chromatographic column;
k) eluting said immunoglobulin from said second packed
chromatographic column with a buffer;
l) reconditioning said second packed chromatographic column with a
buffer;
m) eluting said -lactalbumin from said second packed
chromatographic column with a buffer;
n) reconditioning said second packed chromatographic column with a
buffer;
o) eluting said bovine serum albumin from said second packed
chromatographic column with a buffer; and
p) eluting said lactoferrin from said second packed chromatographic
column with a buffer.
5. The method of claim 4, wherein said second packed
chromatographic column is radial flow column.
6. The method of claim 4, wherein said permeate is combined with
said eluate obtained at step f) to produce a fat substitute.
7. The method of claim 6, wherein said fat substitute comprises
about 60% said eluate and 40% said permeate.
8. The method of claim 4, further comprises combining said
.alpha.-lactalbumin, immunoglobulin, and said bovine serum albumin
to produce an infant formula.
9. The method of claim 8, wherein said infant formula further
comprises at least 25% of said lactoferrin and less than one half
of a percent of .beta.-lactoglobulin.
10. The method of claim 9, wherein said infant formula further
comprises casein hydrolysate, fat, nonfat milk solids,
carbohydrate, minerals, and vitamins.
11. The method of claim 8, wherein said infant formula further
comprises about 43.5% of said .alpha.-lactalbumin eluted from said
second packed chromatography column at step m), about 31.6%
lactoferrin eluted at step p), about 15.4% immunoglobulin eluted at
step k), and about 9.5% bovine serum albumin eluted at step o).
Description
FIELD OF THE INVENTION
The present invention is related to the separation of whey
proteins, particularly to the sequential separation of whey
proteins using chromatography and to food related and
pharmaceutical formulations using separated whey proteins.
BACKGROUND OF THE INVENTION
It is well known that the dry content of cow's milk is about 12.5%
of which 3.4% constitute total proteins, 3.5% comprise fat
components, 4.7% lactose and 0.9% ash. The protein component
consists mainly of casein and whey proteins. Other minor components
include non-proteinaceous nitrogen compounds, protease peptones,
and other minor enzyme proteins.
In the cheese industry, milk proteins are separated into caseins
and whey proteins, mainly by two types of precipitation
techniques--rennet precipitation and acid precipitation. In rennet
precipitation, rennin is added to warm milk (30.degree.-35.degree.
C.). The caseins are precipitated leaving the whey proteins in
solution. This type of whey is referred to as sweet whey. Acid
precipitation is carried out at the isoelectric point of milk which
is 4.7 by using a suitable acid. The whey resulting from acid
precipitation is referred to as acid-whey. The choice of the method
depends on the desired cheese product.
Whey which is a byproduct of the cheese industry has a high
nutritional value because of the many valuable proteins in its
composition. However, until recently, a major portion of
commercially produced whey was discarded, causing major
environmental pollution problems. With the advent of stricter
environmental controls and regulations and the availability of more
recent techniques like membrane separation including
ultrafiltration and reverse osmosis, whey proteins and other
products constituted therefrom have become increasingly important
in satisfying the needs of the pharmaceutical, dietetic and food
industries. Research efforts with varying degrees of success in the
area of the isolation of individual proteins from whey and
formulations constituted therefrom abound in the dairy and related
industries.
The following patents exemplify the various prior art efforts to
isolate individual proteins and other constituents from whey and
food and pharmaceutical products derived therefrom.
U.S. Pat. No. 5,077,067 issued Dec. 31, 1991, to Philippe A.
Thibault discloses a process for the selective and quantitative
removal of lactoglobulins from whey proteins.
U.S. Pat. No. 5,055,558 issued Oct. 8, 1991 to Emilia Chiancone and
Maurizio Gattoni describes a method for the selective extraction of
.beta.-lactoglobulin from whey or milk by subunit exchange
chromatography.
U.S. Pat. No. 4,791,193, issued Dec. 13, 1988, to Shigeo Okonogi et
al., is directed to a method for the preparation of pure
lactoferrin from whey or skim milk.
U.S. Pat. No. 4,668,771, issued May 26, 1987, to Hiroshi Kawakami
et al., provides a method for the isolation and purification of
bovine lactoferrin.
U.S. Pat. No. 4,997,914 issued Mar. 5, 1991 to Hiroshi Kawakami et
al., describes a method for the separation and purification of
lactoferrin by adsorption chromatography.
U.S. Pat. No. 4,820,348 issued Apr. 11, 1989 to Matti Harju is
directed to a chromatographic method for the separation of lactose
from milk.
U.S. Pat. No. 4,446,164 issued May 1, 1984 to Roy A. Brog relates
to milk like compositions constituted from sweet whey base with
additives like soluble proteins, edible vegetable oils, non-fat dry
milk solids, sugar or synthetic sweeteners included therein.
U.S. Pat. No. 5,085,881 issued Feb. 4, 1992, to Hans G. Moeller is
describes a process for separating fractions from dried milk or
milk products for use as food stuffs or food or pharmaceutical
adjuvants.
U.S. Pat. No. 5,093,143 issued Mar. 3, 1992 to Horst Behr and
Friedrich Manz deals with nutrient compositions which simulate milk
and are rich in energy and calcium content but poor in albumin and
phosphorus.
U.S. Pat. No. 4,202,909 issued May 13, 1980 to Harold T. Pederson,
Jr., describes a process for the treatment of whey to produce pure
lactose and salt products.
U.S. Pat. No. 5,008,376 issued Apr. 16, 1991 to Robin C. Bottomley
discloses a process for producing a whey fraction with a high
concentration of alpha-lactalbumin by ultrafiltration
technology.
U.S. Pat. No. 3,969,337 issued Jul. 13, 1976 to Karl Lauer et al.,
discloses a method for the chromatographic fractionation of
whey.
As the foregoing patents and other literature articles demonstrate,
although different laboratory and commercial processes are
available for the separation, removal, concentration, and/or
purification of selected whey proteins, these prior art methods
result in the destruction or disposal of all but one selected
protein from the whey, thereby wasting the other valuable proteins
therefrom. None of these prior art methods achieve the separation
of various proteins from whey in a single process step. It would be
desirable, therefore, to provide a method for the continuous and
sequential separation of various proteins from whey in a one or two
step separation process.
Accordingly, it is an object of the present invention to provide a
separation technique which effects a complete sequential separation
of whey proteins in one or two process steps.
Another object of the present invention is to provide a separation
technique for the sequential and continuous separation of whey
proteins which is suitable for laboratory as well as commercial
applications using radial flow chromatography technology.
Yet another object is to provide different buffers which are mild
enough to use in sequentially separating whey proteins without
denaturing them.
Still another object is to provide a separation technique
applicable for food and pharmaceutical uses of whey proteins.
Another object of the invention is to provide dietary and
pharmaceutical formulations comprising various separated whey
proteins in differing proportions.
Additional objects, advantages and novel features of the invention
will be set forth in part in the description which follows and in
part will become apparent to those skilled in the art upon
examination of the following or may be learned by practice of the
invention. The objects and advantages of the invention may be
realized and attained by means of the instrumentalities and
combinations particularly pointed out in the appended claims.
SUMMARY OF THE INVENTION
To achieve the foregoing and other objectives and in accordance
with the purpose and principles of the invention as set forth
herein, the present invention basically provides a process for the
sequential separation of at least five different proteins from whey
and incorporating these separated whey proteins into pharmaceutical
and food formulations. The process of the invention is directed to
the continuous, sequential separation of whey proteins by
chromatography, comprising adsorbing the proteins in liquid whey on
a suitable separation medium packed in a chromatographic column and
sequentially eluting IgG, .beta.-Lg, .alpha.-La, BSA and
lactoferrin fractions with buffers at suitable pH and ionic
strength. Even though both axial and radial flow chromatography may
be utilized, a horizontal flow column is particularly suitable for
the process of this invention. The whey proteins separated by the
process of the invention include .beta.-lactoglobulin (.beta.-Lg),
.alpha.-lactalbumin (.alpha.-La), bovine serum albumin (BSA),
immunoglobulin (Ig-G) and Lactoferrin (L-Fe). The various
formulations of the invention for dietary or pharmaceutical
applications incorporate these separated proteins in various
proportions.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a graphic representation of the elution pattern of the
various proteins in accordance with this invention.
FIG. 2 presents an elution profile of separated proteins vs.
time.
FIG. 3 represents the elution pattern and the location of peak
4.
DETAILED DESCRIPTION OF THE INVENTION
According to the process of the invention, a sample of a starting
material selected from pasteurized sweet whey, pasteurized acid
whey, non-pasteurized acid whey obtained as a by-product of cheese
manufacture, or whey protein concentrate, prepared from the
pasteurized or non-pasteurized whey by known techniques such as
reverse osmosis (RO) or ultrafiltration (UF) is loaded on to a
chromatographic column, preferably a horizontal flow
chromatographic column, packed with either an acidic or basic,
cationic or anionic resin material such as macro-prep high S or Q.
The whey, concentrated whey or whey protein concentrate may be
subjected to pre-separation procedures such as de-ashing through
electrodialysis or ion exchange, clarification to remove casein
fines, and/or microfiltration for separating colloidal and
suspended particles including fat residues.
The various whey components were then eluted and separated
according to the protocols described in the following examples.
EXAMPLE 1
Sequential Separation Of Sweet Whey Proteins
Commercial whey, a by-product of mozzarella cheese manufacture, was
initially clarified to remove casein fines, centrifuged to remove
milk fat residue, pasteurized at 162.degree. F. for about 18
seconds, and chilled to 40.degree. F. by passing it through HTST
plate heat exchangers. 1000 ml of this skimmed commercial sweet
whey at pH 6.4 and 6.2% total solids, was pH adjusted to 3.8 with
acetic acid at 40.degree. F. The composition of this whey product
used in this experimental example is presented in Table I.
TABLE I ______________________________________ WHEY Components
Percentage Protein Composition
______________________________________ Total solids 6.2
.beta.-lactoglobulin 0.29% Lactose 4.5 .alpha.-lactalbumin 0.13%
Protein 0.8 Serum casein 0.21% Fat 0.08 Immunoglobulin 0.06% Ash
0.77 Lipoprotein 0.06% Lactic Acid 0.05 Bovine serum 0.03% albumin
Lactoferrin 0.02% ______________________________________
The whey was then passed through a 250 ml radial flow
chromatographic column prepacked with a strong S cation exchange
resin and equilibrated with 0.05M acetate buffer at pH 3.8. All the
whey proteins were bound to the resin matrix, and the effluent
containing non-protein components including lactose, minerals,
lactic acid, and non-protein nitrogenous components was allowed to
pass through. The resin with the bound proteins was then washed
with 0.05M acetate buffer at pH 3.8 to a preset UV baseline. The
various bound proteins were then sequentially eluted in accordance
with the following protocol:
Immunoglobulin (IgG) and .beta.-lactoglobulin (.beta.-Lg) were
eluted in sequential order with a buffer at pH 4.0 containing 0.1M
sodium acetate and 0.5M sodium chloride.
The column was then reconditioned and equilibrated with 0.05M
sodium acetate buffer at pH 4.0, to bring the conductivity back to
the base line.
.alpha.-Lactalbumin (.alpha.-La) fraction was eluted with a pH 5.0
buffer containing 0.1M sodium acetate and 0.1M sodium chloride. The
column was again reconditioned with a pH 5.0 buffer containing
0.05M sodium acetate to bring the conductivity back to the
initially established base line. Bovine serum albumin (BSA) was
then eluted with a 0.05M phosphate buffer at pH 7.0. Thereafter,
lactoferrin (LF) was eluted at pH 7.5 with a buffer containing
0.05M sodium phosphate and 0.5M sodium chloride.
The column was again regenerated by washing it with a solution
containing 0.2M sodium hydroxide and 1M sodium chloride, followed
by a wash with a 20% ethanol (EtOH) solution to sterilize the
column and equilibrated with acetate buffer at pH 3.8 for
reuse.
A flow diagram showing the elution protocol is presented in Table
II below.
TABLE II ______________________________________ Flow Diagram Of
Elution Protocol ______________________________________ ##STR1##
##STR2## ##STR3## ##STR4## ##STR5## ##STR6## ##STR7## ##STR8##
##STR9## ##STR10## ##STR11## ##STR12## Repeat Sequence
______________________________________
Fractions of each of the eluted proteins were collected in terms of
elution "peaks" for further separation, concentration, and other
treatment protocols. The elution sequence with the different
protein peaks in terms of their UV absorption at 280 nm is
presented in FIG. 1. Protein identification of each peak was
monitored by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE), as known in the art. Protein recovery
as monitored by bio-rad and gel scan assays at various stages of
the elution scheme is presented in Tables III and IV.
TABLE III ______________________________________ Summary of Bio-Rad
Assay Data ______________________________________ (#0) Column Load
1.0 L treated whey 4.3 g loaded 4.3 mg/ml total protein (#1) Column
Flow-Through 1.65 L 0.3 g (7%) 0.2 mg/ml total protein (#2)
.beta.-Lg + IgG Fraction 2.4 L 2.9 g (67%) 1.2 mg/ml total protein
(#3) .alpha.-La Fraction 1.25 L 0.6 g (14%) 0.5 mg/ml total protein
(#4) BSA Fraction 1.625 l 0.4 g (9%) 0.3 mg/ml total protein (#5)
L-Fe Fraction 0.625 L 0.05 g (1%) 0.09 mg/ml total protein (#6)
Wash 1 1.6 L 0.05 g (1%) 0.03 mg/ml total protein (#7) Wash 2 0.4 L
0.04 g (1%) 0.09 mg/ml total protein Total Recovery = 4.3 g = 100%
accountability ______________________________________
TABLE IV
__________________________________________________________________________
Summary Of Gel Scan Data
__________________________________________________________________________
Gel #1 R2 = 0.98 Column Load 1.0 L treated whey 4.5 g loaded 4.5
mg/ml total protein Approximate % Composition 76% .beta.-Lg No
protein eluted in 17% .alpha.-La flow-through 2% BSA 3% IgG 2%
Other Gel #2 R2 = 0.99 Volume = 2.4 L .beta.-Lg and IgG Fraction
2.9 g recovered (64%) 1.2 mg/ml total protein 94% .beta.-Lg 2% IgG
Gel #3 R3 = 0.993 Volume = 1.25 L .alpha.-La Fraction 0.4 g
recovered (9%) 0.03 mg/ml total protein 94% .alpha.-La Gel #4 R3 =
0.991 Volume = BSA Fraction 0.625 L L-Fe Fraction 0.08 mg/ml total
protein in BSA Fraction 0.13 g (3%) 62% BSA recovery (minimum) 0.05
mg/ml total protein in L-Fe Fraction 0.03 g (1%) 45% L-Fe recovery
(minimum)
__________________________________________________________________________
EXAMPLE 2
An Alternative Protocol For Elution Of Whey Proteins
A 20 liter RFC column was packed with a macro-prep 50 S resin. The
column was then conditioned, equilibrated, loaded, eluted and
reconditioned in exactly the same manner as described in Example 1
above, except that the flow rates, volume of whey loaded on to the
column, flow rates and buffer volumes were varied. Protein elution
peaks were monitored at 280 nm using a uv spectrophotometer. A
graphical trace of the eluted proteins with their relative
concentrations is presented in FIG. 2. The proteins eluted with
their respective percentages of purity are shown in Tables V and
VI.
TABLE V ______________________________________ Protein Yields In
Eluate Fractions (20 L column; Flow Rate 8 L/min; Whey Load 80 L)
Protein Volume Protein Yield (g/L) (L) Load (g) %
______________________________________ Whey Load 8.8 80 704 -- Flow
Through (P-1) 0.9 97 87 12 .beta.-La + IgG (P-2) 2.8 89 249 35
.alpha.-Lactalbumin (P-3) 1.1 94 103 15 BSA (P-4) 1.1 102 112 16
Lactoferrin (P-5) 0.9 45 41 6 Rinse (P-6) 0.8 22 18 3 Stripping
Solution (P-7) 1.2 29 35 5 Protein Recovery: 84%; Protein
Accountability: 92% ______________________________________
TABLE VI ______________________________________ Protein Purity (Gel
Scan) % ______________________________________ .beta.-Lactoglobulin
82 Immunoglobulin 11 .alpha.-Lactalbumin 84 Bovine Serum Albumin 59
Lactoferrin 52 ______________________________________
EXAMPLE 3
Preparation Of An Anionic Exchange Resin Column
A 250 ml RFC column was packed with a strong base, anionic exchange
resin--macro-prep 50 Q--and conditioned with 0.2M NaOH+1M NaCl at a
flow rate of 100 ml/min for 10 minutes. The column was then
equilibrated with 0.01M sodium phosphate at pH 6.90 at a flow rate
of 100 ml/min for 10 minutes. This column was then used to separate
immunoglobulins (IgG) from .beta.-Lactoglobulin eluted as
overlapping peaks from Examples 1 and 2 above. This mixture may be
incorporated into dietary formulations or used for further
separation of the two protein components.
EXAMPLE 4
Separation Of Immunoglobulins (IgG) from .beta.-Lactoglobulin
The eluate represented by peak 2, collected from the fractionated
material from the process described in Example 1, and containing
IgG and .beta.-Lg at pH 4.0, was passed through a 10,000 molecular
weight cut-off UF membranes for concentrating the proteins and for
reducing the buffer salt concentration and thereby, the ionic
strength of the solution. The proteins were further concentrated to
5.times. their initial eluted concentrations and buffer salt
concentrations were reduced to about one-fourth their eluting
concentration by diafiltration with distilled water. The
diafiltered and concentrated protein solution was pH adjusted to
6.9 with a 2.0M solution of NaOH. Two liters of this protein
solution at pH 6.9 was loaded on to the pre-conditioned RFC column
as described in Example 3, at a flow rate of 100 ml/min. The column
was washed with 0.01M sodium phosphate buffer at pH 6.9 to
establish a UV baseline. IgG which did not bind to the resin passed
through the column with the wash and was collected for further
processing. The adsorbed .beta.-Lg was then eluted from the column
with 0.05M sodium citrate buffer at pH 3.0 and collected. The
column was rinsed with distilled water, stripped of residual
proteins with 0.2M NaOH+1M NaCl solution, followed by 20% EtOH and
again reequilibrated with sodium phosphate buffer at pH 6.9, in
preparation for the next cycle.
EXAMPLE 5
Separation And Isolation Of BSA
The eluate represented by peak 4, collected from the fractionated
material from the process described in Example 1, and containing
BSA, and protease peptone at pH 7.0 was concentrated and
diafiltered as described in Example 4, then pH adjusted to 5.5 with
acetic acid. A 250 ml RFC column prepared as described in Example 4
was rinsed with distilled water at a flow rate of 100 ml/min. Two
liters of the protein solution were loaded onto the column as
described earlier. The column was again flushed with distilled
water at a flow rate of 100 ml/min to elute the nonadsorbed
protease peptone and to establish a stable UV baseline. The eluate
containing the protease peptone was collected for further use. The
adsorbed BSA was thereafter eluted with sodium phosphate buffer
containing 0.2M sodium chloride at pH 7.0. FIG. 3 represents the
elution pattern and the location of peak 4.
EXAMPLE 6
Elution And Separation Of .beta.-Lactoglobulin From Liquld Whey
A 250 ml radial-flow chromatographic column packed with a strong
base anionic exchange resin (macro-prep 50 Q) was washed and
regenerated according to manufacturer's instructions. The column
was then equilibrated with 0.05M sodium phosphate (tribasic) at pH
7.5 at a flow rate of 100 ml/min for 10 min. pH ranges of 7.0 to
8.0 did not significantly affect the elution pattern. Two liters of
clarified, skimmed, pasteurized sweet whey from mozzarella cheese
manufacture, chilled to 40.degree. F. were pH adjusted to 8.0 with
5M sodium hydroxide, were circulated through the pre-prepared
column at 75 mils/min for equilibration. Flow rates in the range of
50.gtoreq.100 ml/min were utilized. Then a 1 to 3 liter sample of
whey to be analyzed was loaded on to the column and the column
eluted with the loading buffer--0.05M sodium phosphate at pH 7.5.
Under the conditions utilized, all whey proteins except
.beta.-lactoglobulin are positively charged. .beta.-lactoglobulin
being negatively charged, is bound to and retained by the anionic
exchange resin. The effluent containing non-bound proteins
[.alpha.-Lactalbumin (.alpha.-La), Immunoglobulin (Ig-G), bovine
serum albumin (BSA) and lactoferrin (L-Fe)] was allowed to pass
through the column, collected and stored at 40.degree. F. for
further processing.
The adsorbed B-lactoglobulin was then eluted from the column with a
pH 7.5 buffer containing 0.05M sodium phosphate and 0.5M sodium
chloride. This eluate containing .beta.-lactoglobulin may be
processed further to prepare a shelf stable product in the same
manner as described in Example 8 below.
The column was washed with 1M sodium chloride at a flow rate of 125
ml/min for about four column volumes (2 liters), stripped with 1M
sodium hydroxide at the same flow rate, regenerated with 1M sodium
chloride at a flow rate of 100 ml/min for about five column volumes
(21/2liters) sanitized with 200 ppm sodium hypochlorite at 100
ml/min for about four column volumes (2 liters) and then
equilibrated with the loading buffer in preparation for the next
cycle.
EXAMPLE 7
Elution And Sequential Separation Of Four Proteins From A
Non-.beta.-Lactoglobulin Fraction Of Liquid Whey
The flow-through fraction from Example 6, containing 0.55% protein,
was passed through a 10,000 molecular weight cut-off, spiral
ultra-filtration membrane to a 35% of the original volume, removed
as a permeate for the purpose of partial protein concentration and
also for reduction of soluble salts. This pre-treatment procedure
facilitates the optimum absorption and sequential desorption of
Ig-G, .alpha.-La, BSA and L-Fe protein fractions as outlined in
Example 1. The prepared flow-through was pH adjusted to 3.8 with
acetic acid and 1500 ml sample of it was loaded onto a 250 ml RFC
column packed with a strong S-cationic exchange resin and
pre-equilibrated with 0.05M sodium acetate buffer at pH 3.8.
Washing, sequential elution and regeneration steps as outlined in
Example 1 were followed. The eluted protein fractions were
individually passed through the appropriate molecular weight
cut-off--50000 MW cut-off membrane for Ig-G, L-Fe and BSA and
10,000 MW cut-off membrane for .alpha.-La--to concentrate proteins
and eliminate salt residues. It was then processed further to a
finished product as outlined in Example 8.
EXAMPLE 8
Flow Diagram Showing Preparation Of Final Product ##STR13##
The whey protein fractions or the separated and purified proteins
and the non-proteinaceous eluants may be incorporated into dietary
and pharmaceutical formulations in appropriate proportions. Such
formulations include but are not limited to infant formulas, fat
substitutes, foaming agents, egg white substitutes, animal feed
substitutes and the like.
EXAMPLE 9
Infant Formulas
In the infant formulas constituted in accordance with this
invention, the casein and whey fractions of cow's milk were
modified to achieve a composition simulating human milk to a
significantly larger degree than prior art compositions and
commercial products. The infant formulas of the present invention
contain whey proteins at levels similar to those in human milk.
This was achieved by producing a whey protein ingredient mix
containing the type and ratio of whey proteins of human milk.
Commercially available infant formulas are constituted from whole
cow's milk, mostly because of its availability on a large scale.
Other additives or adjuvants may be included. These formulas are
manufactured either in powder, concentrated or ready to feed
preparation. They consist, for the most part, of non-fat milk
solids, vegetable oils and carbohydrate sweeteners such as lactose,
corn syrup solids and sucrose. These formulas may also be fortified
with vitamin C, vitamin D, iron and fluoride. Table VlI shows the
typical compositions of a few exemplary commercial infant formulas
in comparison to one exemplary formula of the present invention.
Levels of vitamins, minerals and other fortifiers in the
formulation of the present invention are adjusted to simulate human
milk and to meet nutritional requirements of infants.
TABLE VII
__________________________________________________________________________
Composition Range Of Some Commercial Infant Formulas Compared To
One Exemplary Formulation Of The Present Invention Commercial
Infant Formulas (Similac, Alimentum, Good Start, Gerber etc.)
Formula Of Present Invention Per 5 Oz Prepared Per 5 oz Prepared
Nutrients Feed (.about.12.5% solids) Ingredients Feed (.about.12.5%
solids) Ingredients
__________________________________________________________________________
Protein 2.14-2.75 g NFDM*-Casein 1.1-2.50 g Dry or wet mix
hydrolysate-whey of purified, protein composition selected proteins
Fat 5.1-5.54 g veg, coconut, soy, 4.30-6.48 g veg oils, milk fat
palm, safflower, sun flower oil etc. Carbo- 10.2-11.0 g lactose,
sucrose 10.19-10.50 g lactose hydrate Water 133-135 g 15-130 g
Linoleic 850-1600 mg 1200-1300 mg Acid Vit. A 300 IU 300-350 IU
Vit. D 45-60 IU 50-60 IU Vit. E 2.0-3.0 IU 2.2-2.7 IU Vit. C 9 mg
7-9 mg Vit. K 8-15 mcg 8-10 mcg Vit. B1 60-100 mcg 25-100 mcg Vit.
B2 90-150 mcg 50-150 mcg Vit. B6 60-75 mcg 20-60 mcg Vit. B12
0.22-0.45 mcg 0.10-0.25 mcg Niacin 750-1350 mcg 300-1100 mcg Folic
Acid 9-15 mcg 7-15 mcg Pantothenic 450-750 mcg 330-450 mcg Acid
Biotin 2.2-4.5 mcg 2-4 mcg Choline 8-16 mg 10-16 mg Inositol 4.7-18
mg 4.5-5.5 mg Calcium 64-105 mg 47-73 mg Phosphorus 36-75 mg 21-56
mg Magnesium 6.0-7.5 mg 4.4-6.0 mg Iron 0.5-1.8 mg 0.04-1.8 mg Zinc
0.75 mg 0.25-0.75 mg Manganese 5-30 mcg 5-10 mcg Copper 75-90 mcg
75-90 mcg Iodine 8-15 mcg 9-12 mcg Sodium 24-44 mg 25-40 mg
Potassium 98-118 mg 75-110 mg Chloride 59-80 mg 59-80 mg
Cholesterol -- 18-25 mg
__________________________________________________________________________
*NFDM-non-fat dry milk solids
However, due to the large differences in the protein compositions
of cow's milk and human milk, some infants show different degrees
of intolerance to cow's milk and food formulas constituted
therefrom. A comparison of the composition of cow's milk with that
of human milk is presented in Table VII and a comparison of their
protein content is presented in Table IX.
TABLE VIII ______________________________________ Comparison of
Cow's Milk With Human Milk Per 100 g Cow Human
______________________________________ Water g. 89.99 87.5 Food
Energy kcal. 61 70 Protein (N .times. 6.38 ) g. 3.29 1.03 Fat g.
3.34 4.38 Carbohydrate (total) g. 4.66 6.89 Fiber g. 0 0 Ash g.
0.72 0.2 Minerals Calcium mg. 119 32 Iron mg. 0.05 0.03 Magnesium
mg. 13 3 Phosphorus mg. 93 14 Potassium mg. 152 51 Sodium mg. 49 17
Zinc mg. 0.38 0.17 Vitamins Ascorbic Acid mg. 0.94 5.00 Thiamin mg.
0.038 0.014 Riboflavin mg. 0.162 0.036 Niacin mg. 0.084 0.177
Pantothenic Acid mg. 0.314 0.223 Vitamin B.sub.6 mg. 0.042 0.011
Folic Acid mcg. 5 5 Vitamin B.sub.12 mcg. 0.357 0.045 Vitamin A
I.U. 126 241 Cholesterol mg. 14 14
______________________________________
TABLE IX ______________________________________ Protein Composition
Of (Cow & Human Milk) g/100 g Protein Cow Human
______________________________________ Casein (total) 2.6 0.32
.beta.-Lactoglobulin 0.32 Negligible .alpha.-Lactalbumin 0.12 0.28
Serum albumin 0.04 0.06 Lysozyme Negligible 0.04 Lactoferrins 0.02
0.20 Immunoglobulins 0.07 0.10
______________________________________
A ratio of the various whey proteins between cow's and human milk
is presented in Table X.
TABLE X
__________________________________________________________________________
Ratio Of Various Whey proteins in Cow's & Human Milk Mix
Protein Cow's Milk (g/100 g) % (1) Human Milk % Ratio H/C (2) 1
.times. 2 Composition
__________________________________________________________________________
.alpha.-La 0.12 48 0.28 43.75 2.33 1.10 43.5 L-Fe 0.02 8 0.20 31.25
10 0.80 31.6 IgG 0.07 28 0.10 15.63 1.43 0.39 15.4 BSA 0.04 16 0.06
9.37 1.5 0.24 9.5
__________________________________________________________________________
As shown in the foregoing tables, cow's milk contains 3.3% protein
while human milk has only 1%. Caseins are the major protein
components in cow's milk (about 77% of total protein) whereas human
milk contains a high ratio of whey proteins to caseins (about 2:1).
.beta.-Lactoglobulin concentration in cow's milk is the highest of
the whey proteins while it is negligible in human milk similarly,
lactoferrin is ten times higher in concentration in human milk than
in cow's milk. Immunoglobulin and serum albumin concentrations are
about 1.5 times higher in human milk than in cow's milk.
In the infant formula of the present invention, lactose and fat
levels are adjusted to simulate human milk. Vegetable fat replaces
butter fat. Casein to whey ratio is also reduced to simulate human
milk. Other additives and supplements such as vitamins, taurine,
and minerals may be included if desired. The total solute load is
reduced to the level found in human milk.
To achieve this objective, whey protein fractions obtained from the
fractionation and elution in accordance with the process of this
invention, were first combined in the ratio shown in Table XI
below.
TABLE XI ______________________________________ Dietary Formulation
A ______________________________________ .alpha.-Lactalbumin
fraction 43.5% Lactoferrin fraction 31.6% Immunoglobulin fraction
15.4% Bovine serum albumin fraction 9.5%
______________________________________
The above whey protein mix was then incorporated into a human
milk-like formulation with the composition shown in Table XII.
TABLE XII ______________________________________ Infant Formula
Liquid formula Dry Formula Base Ingredient (g/100 g) (g/6 oz
liquid) ______________________________________ Water 87.20 0
.beta.-Casein 0.28 0.504 .kappa.-Casein 0.04 0.072 ##STR14##
##STR15## 1.152 Lysozyme 0.04 0.072 Lactoperoxidase 750 (activity)
1350 Fat 4.5 8.1 Lactose 7.0 12.6 Ash 0.2 0.36
______________________________________
The liquid mix prepared according to the above composition
contained about 2% total solids, (of which salts from the eluting
buffers comprise 90% and total proteins comprise about 10%), and
98% water. This liquid mix was then concentrated through a 10,000
molecular weight cut-off, spiral ultrafiltration membrane to 5-15%
total proteins, followed by diafiltration with distilled water at
0.5-1.0.times. to remove remaining salt residues. This formulation
may be further concentrated by processes normally utilized in the
treatment of labile proteins, such as ultrafiltration, reverse
osmosis, freeze drying, freeze concentration, spray drying and the
like or any combination thereof. The formulations of this invention
may be further fortified with suitable additives and fortifiers.
Such additives and fortifiers include but are not limited to nonfat
milk solids, vegetable solids, carbohydrate sweeteners, minerals
and vitamins. The solid composition of one exemplary formulation of
the present invention is presented in Table XIII.
TABLE XIII ______________________________________ Solid Composition
Of One Exemplary Formulation Of The Present Invention Ingredients
gm/16 oz of Formula Powder ______________________________________
Proteins: Casein Hydrolysate 11.00 .alpha.-Lactalbumin 9.63 Bovine
Serum Albumin 2.06 Lactoferrin 6.88 Immunoglobulins 3.44 Lysozyme
1.38 Fat Coconut Oil 53.56 Sunflower Oil 46.08 Corn Oil 36.79
Butter Fat 14.10 Carbohydrate: 236.95 Lactose Moisture (Water
Content): 15.20 Linoleic Acid: 2.41 Vitamins: Vit. A 8288 (IU) Vit.
D 1720 (IU) Vit. E (tocopherol) 86 (IU) Vit. K 0.0003 Vit. B1
0.0018 Vit. B2 0.0012 Vit. B6 0.0004 Vit. B12 0.0000002 Vit. C
(ascorbic acid) 0.17 Niacin 0.006 Folic Acid 0.0001 Pantothenic
Acid 0.008 Biotin 0.0001 Choline 0.34 Inositol 0.18 Minerals:
Calcium 1.10 Phosphorus 0.48 Magnesium 0.10 Iron 0.001 Zinc 0.006
Manganese 0.0003 Copper 0.003 Iodine 0.0003 Sodium 0.59 Potassium
1.76 Chloride 2.06 Cholesterol 0.48
______________________________________
EXAMPLE 10
Formulations As Fat Substitutes
.beta.-lactoglobulin exhibits high water-binding qualities and
.alpha.-lactalbumin increases viscosity and also absorbs a high
content of fats and oils because of these properties, a combination
of these proteins would lend itself to use as fat substitutes. This
product when incorporated in appropriate proportions into some food
products improves the quality and characteristics of the product
and may be used as a fat substitute.
.beta.-lactoglobulin elutes, and along with IgG, as fraction 2 in
the process of the present invention and .alpha.-lactalbumin is
eluted in fraction 3. IgG is separated from the
.beta.-lactoglobulin fraction as described in Example 7 and is
mixed with the .alpha.-lactalbumin fraction in a 60 and 40% ratio.
The mix of the two proteins (.beta.-La+.alpha.-La) is passed
through a 10,000 molecular weight cut-off ultra-filtration membrane
at 40.degree. F. with a differential pressure of 10 psi and the
permeate, consisting of water and soluble salts, is removed until a
40-50% total solids concentration is achieved. This is followed by
a diafiltration at 0.5.times. with distilled water to remove
remaining salt residues. The concentrated and purified mix thus
obtained may be frozen, freeze-dried, chilled or dehydrated for
further use. Other additives such as flavor enhancers, vitamins and
sweeteners may be included in these formulations as desired.
It has thus been shown that the present invention provides a method
for the continuous and sequential separation of whey proteins and
formulation of the eluted fractions which may be used as food
additives or substitutes.
The foregoing description of the preferred embodiments of the
subject invention have been presented for purposes of illustration
and description and for a better understanding of the invention. It
is not intended to be exhaustive or to limit the invention to the
precise form disclosed; and, obviously, many modifications and
variations are possible in the light of the above teaching. The
particular embodiments were chosen and described in some detail to
best explain the principles of the invention and its practical
application thereby to enable others skilled in the relevant art to
best utilize the invention in various embodiments and with various
modifications as are suited to the particular use contemplated. It
is intended that the invention be defined by the claims appended
hereto.
* * * * *